Neoantigen-specific T cell help outperforms non-specific help in multi-antigen DNA vaccination against cancer

CD4+ T helper antigens are essential components of cancer vaccines, but the relevance of the source of these MHC class II-restricted antigens remains underexplored. To compare the effectiveness of tumor-specific versus tumor-unrelated helper antigens, we designed three DNA vaccines for the murine MC-38 colon carcinoma, encoding CD8+ T cell neoantigens alone (noHELP) or in combination with either “universal” helper antigens (uniHELP) or helper neoantigens (neoHELP). Both types of helped vaccines increased the frequency of vaccine-induced CD8+ T cells, and particularly uniHELP increased the fraction of KLRG1+ and PD-1low effector cells. However, when mice were subsequently injected with MC-38 cells, only neoHELP vaccination resulted in significantly better tumor control than noHELP. In contrast to uniHELP, neoHELP-induced tumor control was dependent on the presence of CD4+ T cells, while both vaccines relied on CD8+ T cells. In line with this, neoHELP variants containing wild-type counterparts of the CD4+ or CD8+ T cell neoantigens displayed reduced tumor control. These data indicate that optimal personalized cancer vaccines should include MHC class II-restricted neoantigens to elicit tumor-specific CD4+ T cell help.


Figure S1 .
Figure S1.Verification of vaccine constructs.(A-B) HEK293T cells were transfected with a GFPencoding plasmid in combination with the indicated vaccine-encoding plasmids.Transfected cells were identified by GFP-expression, and a C-terminal HA-tag allowed detection of multi-antigen vaccine proteins.(A) The fraction (%) of HA-positive cells among transfected (GFP + ) cells, and (B) their mean fluorescence intensity (MFI) were determined by flow cytometry.The MFI of mock-transfected cells is not shown as these cells -not having been transfected with a plasmid encoding an HA-tagged protein -do not have detectable expression of the HA-tag above background.Bars and whiskers represent means and standard errors (SEM) of triplicates, respectively.(C-D) Western blot detection of HA-tagged (C) noHELP, neoHELP, uniHELP or (D) neoHELP, neoHELP_CD4wt or neoHELP_CD8wt in HEK293T cells transfected with the indicated vaccine-encoding plasmids.(E) Recognition of OVA antigen on transfected B16-F10 cells by H-2K b /OVA-specific CD8 + T-cell hybridoma B3Z.β-galactosidase expression in B3Z cells is controlled by NFAT, allowing detection of TCR-mediated activation by color conversion of CPRG substrate, which results in increased optical density (OD) at 594nm.Bars and whiskers represent means and standard errors (SEM) of triplicates, respectively.All experiments were performed twice, and one representative experiment is shown.

Figure S2 .
Figure S2.Gating strategy for flow cytometry analysis.(A-B) Gating strategy for spleen-derived Tlymphocytes for ICS.(A) Live CD4 + and CD8 + T-cells were gated after which (B) TNF-, IFN-γ-and IL-2positive cells were selected.(C-E) Gating strategy for antigen-specific peripheral blood-and spleenderived T-lymphocytes for surface marker analysis.(C) Gating of live CD8 + T-cells was followed by (D) selection of tetramer-positive cells.(E) Subsequently, cells positive for specific surface markers were identified.

Figure S3 .
Figure S3.Antigen-specific CD4 + and CD8 + T-cell responses in the spleen of mice.Mice were vaccinated with the indicated vaccines three times, at three-week intervals.Ten days after the final vaccination, spleen cells were cultured with dendritic cells loaded with indicated (A-C) peptide pools or (D-F) individual peptides for 5 hours, and analyzed by intracellular cytokine staining (ICS).IL-2-, TNF-

Figure S4 .
Figure S4.Tumor growth upon depletion of CD4 and CD8 responses.Mice (10 mice per group) were vaccinated three times, at 3-week intervals, with the indicated vaccines.Selected groups were depleted of CD4 + or CD8 + T-cells by three injections of 100 μg of either α-CD4 (clone GK1.5) or α-CD8 (clone 2.43) on days 57, 61 and 64 post primary vaccination.On day 63 post primary vaccination, mice were injected subcutaneously with 300.000MC-38 colon carcinoma cells.Lengths and widths of the tumors were measured multiple times a week with a digital caliper to calculate tumor volume.Mice were removed from the experiment if the tumor exceeded 1000 mm 3 or if an ulcer occurred.Mice were vaccinated with (A) saline solution (mock), uniHELP or neoHELP (B) neoHELP vaccines carrying the wild-type counterparts of either the CD4 (neoHELP_CD4wt) or CD8 (neoHELP CD4wt) T-cell antigens.Additional groups of mice vaccinated with (C) uniHELP and (D) neoHELP underwent CD4 + or CD8 + Tcell depletion around the time of tumor challenge.Tumor volumes of individual mice are plotted, and the numbers of tumor-free mice at day 50 post challenge are indicated at the bottom right of each graph.
cell epitopes in boldface, mutated residues underlined.